Arginine Metabolism: Enzymology, Nutrition, and Clinical Signiﬁcance

1,2 Enzymes of the L-Arginine to Nitric Oxide Pathway Dennis J. Stuehr3 Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 Downloaded from https://academic.oup.com/jn/article/134/10/2748S/4688471 by guest on 30 September 2021 ABSTRACT L-Arginine is the biological precursor of nitric oxide (NO), which serves as an important signal and effector molecule in animals. This review summarizes some structure-function aspects of the mammalian nitric oxide synthases, which are enzymes that catalyze the oxidation of L-arginine to NO and L-citrulline. These include aspects related to: 1) the chemical transformations of L-arginine during enzyme catalysis, 2) binding of L-arginine or its structural analogs to the nitric oxide synthases, and 3) how L-arginine levels may affect product formation by the nitric oxide synthases and how this can be modulated by structural analogs of L-arginine. J. Nutr. 134: 2748S–2751S, 2004.

KEY WORDS: ● L-arginine ● nitric oxide ● ﬂavoprotein ● reactive oxygen species ● hemeprotein

The widespread interest in nitric oxide (NO) biology can biopterin (H4B) is also a required cofactor, and it is tightly be traced back to nutrition and toxicology research conducted bound in NOS next to the heme (10,11). H4B has structural in the early 1980s that established that mammals are indeed and redox roles in the NOS (11,12). The Ks values for Arg and 4 capable of NO biosynthesis (1,2). L-Arginine (Arg) was sub- NOHA binding to NOSs range from 1 to 20 ␮mol/L (6). The sequently found to be the precursor of NO and related N- electron transfer reactions of NOS are regulated by a calcium- oxides (nitrite, nitrate) that are produced by mammals (3–5). binding protein (calmodulin), which can bind to some NOSs This represents a new metabolic pathway for Arg and is of in a reversible manner (6). This enables cells to couple their current interest because of the tremendous biological and NO synthesis to changes in intracellular calcium ion concen- medical importance of NO. tration. Calmodulin binds tightly to other NOSs at all cellular calcium ion concentrations; thus, their NO synthesis is regu- Some general properties of the NO synthases lated primarily through control of gene expression (13). A family of enzymes called the NO synthases (NOSs, EC 1.14.13.39) catalyze the oxidation of Arg to NO and L-citrul- Arg binding site in NOS line, with NADPH and O2 serving as cosubstrates (6). The NOSs first hydroxylate a terminal guanidino nitrogen of Arg Crystal structures of NOS heme domains that contain to generate N-hydroxy-L-arginine (NOHA) as an enzyme- bound Arg or NOHA show in detail how either of these bound intermediate. NOHA is then oxidized further by the substrates bind in the enzyme active site (14–16,10). There is enzyme to generate NO plus L-citrulline (Fig. 1). Three re- a funnel-shaped entrance to the enzyme active site that allows lated NOSs are expressed in mammals, along with several substrate and dioxygen access to the heme. In general, NOS splice variants (7). NOS-like enzymes are also coded for in the binds Arg or NOHA in a similar extended conformation, with genomes of most life forms, including bacteria (8,9). The the guanidino (or hydroxyguanidino) moiety located above mammalian NOSs have a similar structure and composition. the heme iron and the amino acid portion held away from the They are all homodimeric, heme-containing flavoproteins. heme (Fig. 3). The location of the guanidino group above the The NOS flavins transfer NADPH-derived electrons to the heme iron is consistent with the heme iron being the site heme (Fig. 2). This enables the heme to bind and activate where dioxygen binds and becomes activated in NOS, such dioxygen in both steps of NO synthesis. 6-(R)-Tetrahydro- that the resulting heme iron-oxy species can react directly with the guanidino moiety. Arg and NOHA are held in place by hydrogen bonds that form between conserved residues of 1 Prepared for the conference “Symposium on Arginine” held April 5–6, 2004 NOS and both the amino acid and guanidino ends of the in Bermuda. The conference was sponsored in part by an educational grant from substrates. Several of the binding residues are located in a Ajinomoto USA, Inc. Conference proceedings are published as a supplement to substrate-binding helix of NOS (16). The substrate hydrogen- The Journal of Nutrition. Guest Editors for the supplement were Sidney M. Morris, Jr., Joseph Loscalzo, Dennis Bier, and Wiley W. Souba. bonding network extends into the H4B binding site, consis- 2 This work was supported in part by the National Institutes of Health, Grants tent with H4B and Arg binding to NOS in a cooperative CA53914 and GM51491. manner (12) and with the finding that point mutations in the 3 To whom correspondence should be addressed. E-mail: [email protected]. 4 Abbreviations used: Arg, L-arginine; H4B, 6-(R)-tetrahydrobiopterin; NO, nitric H4B binding site of NOS often lower the binding affinity oxide; NOHA, N-hydroxy-L-arginine; NOS, nitric oxide synthase (EC 1.14.13.39). toward Arg (17). The ␣ amino groups of Arg and NOHA also

2748S NITRIC OXIDE PATHWAY 2749S

FIGURE 1 The two reactions of NO synthesis as catalyzed by NOS. The NADPH and oxygen requirements of each reaction are shown. Downloaded from https://academic.oup.com/jn/article/134/10/2748S/4688471 by guest on 30 September 2021 hydrogen bond with a heme propionate group, but the func- tional signiﬁcance of this interaction is unclear. Point mutagenesis studies demonstrate the relative importance of some of the NOS substrate binding residues for Arg binding (18– 20). In particular, there is a conserved glutamic acid whose side-chain carboxylate forms hydrogen bonds with two of the guanidino nitrogens of Arg or NOHA. Removing this interaction by point mutagenesis completely disables Arg binding to NOS. Mutational substitution at other residues involved in Arg or NOHA binding typically has a less drastic effect.

Effects of Arg binding on NO synthase Arg binding causes a number of observable changes in NOSs. It stabilizes the NOS ferric heme iron in a high-spin electronic state, which is typically manifested by the shifting of the heme Soret absorbance band to a lower wavelength (21). Arg binding increases NOS affinity for H4B, consistent with their binding in a cooperative manner (12). Arg binding increases the reduction potential of the NOS heme iron (22). This is important for some NOSs because it makes their heme reduction by the flavins thermodynamically favorable (Fig. 4). Arg binding also stabilizes the dimeric structure of some NOSs (23,24), and stabilizes heme-NO complexes that form during NOS catalysis (25,26). FIGURE 3 Arg and NOHA binding in inducible NOS. The crystal Substrate analogs of Arg structure data indicate how Arg (top) or NOHA (bottom) binds within the active site of NOS in relation to the enzyme heme and H4B groups. Various Arg derivatives can serve as substrates for NOS, Protein residues that make hydrogen bond contacts with Arg or NOHA including non–amino acid analogs (Fig. 5) (27,28). In general, are indicated, with the hydrogen bonds shown as thin lines. Data are NOSs display a lower binding affinity toward these analogs, from Crane et al. (10) and Crane et al. (14). show isoenzyme selectivity, and have slower Vmax values re- garding NO synthesis. The N-hydroxyguanidine analogs typically bind more tightly than the guanidine analogs and sup- port greater NO synthesis. However, NO synthesis from the guanidine analogs is likely compromised due to dissociation of the N-hydroxyguanidine intermediate from NOS during catalysis. These data indicate that NOS interactions with the

FIGURE 2 Mechanism of oxygen activation by the NOS heme. FIGURE 4 Thermodynamic profile for heme reduction in inducible The ferric heme first receives an electron from the FMN hydroquinone NOS. Estimated reduction potentials for NADPH and the NOS flavopro- (FMNH2) that is located in the NOS flavoprotein domain. This enables tein (FADH/FMNH2) are indicated. The right-hand portion of the figure dioxygen to bind, forming the ferric-superoxy species (I). This species shows how bound Arg or H4B increases the reduction potential of the then receives an electron from tetrahydrobiopterin (H4B) to generate ferric heme iron. Electron transfer toward a more positive potential is heme peroxo (II) and perferryl (III) species that are thought to react with favored; arrows indicate the direction of electron transfer. Data are from Arg or NOHA. Presta et al. (22). 2750S SUPPLEMENT Downloaded from https://academic.oup.com/jn/article/134/10/2748S/4688471 by guest on 30 September 2021

FIGURE 7 Effect of some NOS inhibitors on the thermodynamics of neuronal NOS heme reduction. Estimated reduction potentials for NADPH and the NOS ﬂavoprotein (FADH/FMNH2) are indicated. The right-hand portion of the ﬁgure compares how bound Arg and Arg analog inhibitors poise the reduction potential of the ferric heme iron. Electron transfer toward a more positive potential is favored; arrows indicate the direction of electron transfer. Data are from Presta et al. (22).

and subsequently will release reactive oxygen species such as FIGURE 5 Arg analogs that are substrates for NOS. Structures superoxide and H2O2 (31,32). The frequency and biological are shown with the corresponding Km and relative Vmax values for NO synthesis. Charges are not shown. Data are from Dijols (28). effect of this process are under scrutiny. An active NOS operating at a subsaturating Arg concentration can generate NO and superoxide at the same time. These products can amino acid portion of Arg are not essential for enzyme catal- conceivably react to generate the cellular toxin peroxynitrite ysis. (33,34). Certain Arg analogs can inhibit the production of reactive Nonsubstrate analogs of Arg oxygen species by NOS, whereas others either allow it or A great number of Arg derivatives are not substrates for enhance it (35,36). Some of the analogs that inhibit reactive NOS and act instead as competitive inhibitors or in some cases oxygen species production by NOS act by lowering the heme as mechanism-based inactivators (29). Many of these com- midpoint reduction potential, rendering the transfer of elec- pounds no longer contain the guanidino moiety, which is trons from the NOS flavins thermodynamically unfavorable essential for NO synthesis. In some cases, the NOS binding (Fig. 7) (22). This represents one strategy to control produc- affinity toward these analogs is greater than that toward Arg tion of reactive oxygen species by NOS under conditions of itself. Figure 6 depicts the structures of some representative low Arg concentration. analogs. NOS crystal structures that contain some of the bound inhibitors are available (30). Regulating Arg availability for the NOSs In some cells, the expression of cationic amino acid trans- Consequences of suboptimal Arg concentration porters is increased coincident with NOS expression (37,38). At nonsaturating Arg concentrations, a calmodulin-bound Arginase may also be expressed and typically is the chief NOS will continue to bind and activate dioxygen at its heme competitor for Arg in cells and tissues (39–41). In certain environments, arginase expression can deplete Arg levels to the point where NO synthesis by NOS is compromised (42,43). Citrulline can be converted to Arg in many tissues, and this may provide a significant source of Arg in some cases (44). Endogenous methylarginines are also present in tissues at concentrations that may inhibit Arg binding to NOS (45–47). Arg demethylase enzymes may serve to regenerate small amounts of Arg (48). Factors regulating Arg availability are discussed in detail in some of the other papers of this series.

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